Extracellular L-glutamate oxidase ofStreptomyces sp. Z-11-6: Obtainment and properties

Mutagenesis induced with nitrous acid and subsequent selection allowed a genetically stable mutant strain,Streptomyces sp. Z-11-6, to be obtained, whose L-glutamate oxidase activity was 40-fold higher than that of the original natural isolate and was as great as 1.6–1.8 units/ml of culture liquid. A procedure for the isolation and purification of the enzyme was developed; the biochemical properties of the enzyme were studied. Out of 20 amino acids tested (including D-glutamate), the glutamate oxidase fromStreptomyces sp. Z-11-6 was active only with L-glutamate. This allows the concentration of L-glutamate to be determined in the presence of other amino acids. Calcium chloride at a concentration of 0.1–0.5% promoted the secretion of the extracellular glutamate oxidase.     

Streptomyces sp. Z-11-6--a new producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254 Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep-mutagenesis and selection procedure allowed a genetically stable strain, Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

Streptomyces sp. Z-11-6, a novel producer of extracellular L-glutamate oxidase

Glutamate oxidase activity was studied in 1254Streptomyces strains isolated from the zonal soils of various regions of Russia and other countries. Seven strains proved to be producers of extracellular L-glutamate oxidase. The most active producer strain was identified, and the conditions of enzyme biosynthesis were optimized. A multistep mutagenesis-selection procedure allowed a genetically stable strain,Streptomyces sp. Z-11-6, to be obtained, whose glutamate oxidase activity was 40 times higher than that of the original natural isolate.     

Properties and prospects of practical use of extracellular L-glutamate oxidase from Streptomyces sp. Z-11-6

The properties of extracellular L-glutamate oxidase, isolated and purified from Streptomyces sp. Z-11-6 (specific activity, 50.8 U/mg protein; yield, 40%), were studied. A photometrical method of determination of activities of alanine- and aspartate aminotransferases, based on the use of the L-glutamate oxidase and peroxidase, has been developed. This method is sufficiently sensitive to be used for the determination of aminotransferase activities in biological fluids. The presence of other amino acids did not interfere with the analysis and had no effect on the results of determination.     

A novel L-glutamate oxidase from Streptomyces endus. Purification and properties

A new flavoenzyme using molecular oxygen to oxidize L-glutamic acid has been purified to homogeneity, as judged by polyacrylamide gel electrophoresis, from the culture medium of Streptomyces endus. Hydrogen peroxide, 2-oxoglutaric acid and ammonia are formed as products. Among 25 amino acids tested including D-glutamic acid, L-glutamine and L-aspartic acid, only L-glutamic acid is converted. The molecular mass of the enzyme was estimated to be about 90 kDa by gel chromatography and 50 kDa by SDS/PAGE. The subunit contains 1 molecule noncovalently bound FAD. The absorption spectrum shows maxima at 273, 355 and 457 nm and the isoelectric point is at pH 6.2. The Km value for L-glutamic acid in air-saturated phosphate pH 7.0 was estimated to be 1.1 mM, the Km for oxygen was calculated to be 1.86 mM at saturating concentration of L-glutamic acid. The enzymic reaction is inhibited by Ag+ and Hg2+ ions. The enzyme described here distinctly differs from two microbial L-glutamate oxidases purified hitherto, with regard to extremely high substrate specificity and to the subunit structure.     

Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6

L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha2beta2gamma2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha2beta2gamma2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.     

Extracellular tyrosinase from Streptomyces sp. KY-453: purification and some enzymatic properties

A strain of Streptomyces isolated from soil was found to produce a large amount of tyrosinase (monophenol, dihydroxy-L-phenylalanine: oxygen oxidoreductase: EC 1.14.18.1) extracellularly. The enzyme was purified from the culture filtrate about 550-fold by a series of column chromatographies on Duolite A-2 and CM-cellulose and gel filtration on Sephadex G-100. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the hydroxylation of monophenols and the oxidation of diphenols and was most active at pH 6.8 with dihydroxy-L-phenylalanine (L-DOPA) as the substrate. It was inhibited by kojic acid, diethyldithiocarbamate, and inhibitors obtained from micro-organisms. The isoelectric point of the enzyme was 9.9, and the molecular weight was estimated to be 36,000 by gel filtration on Sephadex G-100 and 29,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, which suggests that the enzyme is a monomer. Metal analysis by atomic absorption spectroscopy indicated that the enzyme contains nearly 1 gram atom of copper per mol.     

Enhancement of L-glutamate oxidase production in liquid fermentation of Streptomyces sp. by calcium addition

Summary The effect of calcium ions on production of extracellular L-glutamate oxidase (GluOx) in liquid fermentation of Streptomyces sp. N1 was investigated. By supplementing a relatively large amount of Ca²⁺ (15–40 mM) to the medium, the GluOx production was significantly enhanced, although the microbial growth was inhibited to some degree. For the first time, the highest production (i.e. 6.5 U/ml) and productivity (i.e. 0.25 U/ml/h) of GluOx as ever reported (i.e., 1.2 U/g medium after 144 h solid state fermentation and 0.59 U/ml after 96 h liquid fermentation) were achieved with addition of 30 mM Ca²⁺.     

Properties of and Prospects for Practical Use of Extracellular L-Glutamate Oxidase of Streptomycessp. Z-11-6

The properties of extracellular L-glutamate oxidase isolated and purified from Streptomycessp. Z-11-6 (specific activity, 50.8 U/mg protein; yield, 40%) were studied. A photometric method of determination of activities of alanine and aspartate aminotransferases based on the use of L-glutamate oxidase and peroxidase has been developed. This method is sufficiently sensitive to be used for determining aminotransferase activities in biological fluids. The presence of other amino acids does not interfere with the analysis and has no effect on the results of determination.     

Characterization of cholesterol oxidase from a marine Streptomyces sp. and its cytotoxicity

A marine actinobacterial strain (designated as AKHSS) capable of producing cholesterol oxidase on the enzyme indicator plates was identified as Streptomyces sp. The cell-free lysate of the strain was used for monitoring the production of cholesterol oxidase and the maximal enzyme yields were recorded at 72 h post inoculation. The cholesterol oxidase was purified using polyethylene glycol 4000 precipitation, diethylaminoethyl Sephacel anionic column chromatography and Superdex-200 gel filtration to near homogeneity. Through electron-spray ionization mass spectrometry, molecular mass of the purified enzyme was recorded as 42.84 kDa. The optimum pH of the enzyme was found to be 9 and it was stable up to 60 °C. Metal salts like MgSO₄ and ZnSO₄ stimulated the enzyme activity. The V_max and K_m of the purified enzyme with cholesterol as substrate were found to be 1.22 μmoles/min/mL and 0.54 mM respectively. The enzyme showed significant cytotoxicity on breast (MCF-7), nasopharyngeal (KB) and ovarian (OVCAR) cancer cell lines at very low concentrations ranging from 0.093 to 0.14 μM, as evident from MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell viability assay. Besides, the enzyme exhibited relatively less cytotoxicity on primary mouse embryonic fibroblast (3T3) cells. Thus, cholesterol oxidase of Streptomyces sp. AKHSS could be a potential anticancer agent.     

Purification and properties of mycodextranase from Streptomyces sp. J-13-3.

An enzyme hydrolyzing nigeran (alternating alpha-1,3- and alpha-1,4-linked glucan) was purified from the culture filtrate of Streptomyces sp. J-13-3, which lysed the cell wall of Aspergillus niger, by percipitation with ammonium sulfate and column chromatographies on DEAE-Sephadex A-50, CM-Sephadex C-50, chromatofocusing, and Sephadex G-100. The final preparation was homogenous in polyacrylamide gel electrophoresis (PAGE). The molecular weight of the enzyme was 68,000 by SDS-PAGE and gel filtration. The optimum pH and temperature for the enzyme activity were 6.0 and 50 degrees C, respectively. The enzyme was stable in the pH range from 6.0 to 8.0 and up to 50 degrees C. The enzyme activity was inhibited significantly by Hg+, Hg2+, and p-chloromercuribenzoic acid. The Km (mg/ml) for nigeran was 3.33. The enzyme specifically hydrolyzed nigeran into nigerose and nigeran tetrasaccharide by an endo-type of action, indicating it to be a mycodextranase (EC 3.2.1.61) that splits only the alpha-1,4-glucosidic linkages in nigeran.     

Production and characterization of thermostable xylanase and pectinase from Streptomyces sp. QG-11-3

Streptomyces sp. QG-11-3, which produces a cellulase-free thermostable xylanase (96 IU ml⁻¹) and a pectinase (46 IU ml⁻¹), was isolated on Horikoshi medium supplemented with 1% w/v wheat bran. Carbon sources that favored xylanase production were rice bran (82 IU ml⁻¹) and birch-wood xylan (81 IU ml⁻¹); pectinase production was also stimulated by pectin and cotton seed cake (34 IU ml⁻¹ each). The partially purified xylanase and pectinase were optimally active at 60°C. Both enzymes were 100% stable at 50°C for more than 24 h. The half-lives of xylanase and pectinase at 70, 75 and 80°C were 90, 75 and 9 min, and 90, 53 and 7 min, respectively. The optimum pH values for xylanase and pectinase were 8.6 and 3.0, respectively, at 60°C. Xylanase and pectinase were stable over a broad pH range between 5.4 and 9.4 and 2.0 to 9.0, respectively, retaining more than 85% of their activity. Ca²⁺ stimulated the activity of both enzymes up to 7%, whereas Cd²⁺, Co²⁺, Cr³⁺, iodoacetic acid and iodoacetamide inhibited xylanase up to 35% and pectinase up to 63%; at 1 mM, Hg²⁺ inhibited both enzymes completely. Journal of Industrial Microbiology & Biotechnology (2000) 24, 396–402. Received 29 September 1999/ Accepted in revised form 02 February 2000     

链霉菌Str s-2产木聚糖酶的条件及部分性质研究

通过碳氮源对链霉菌Str s-2产胞外木聚糖酶活性的影响,得出其适宜培养基为(g/L)：含半纤维素20,(NH4)2SO4 4．0,KH2PO4 1．0,MgSO4-7H2O 0．5,NaCl 0．3,CaCO3 1．0。用DNS法研究了该酶的性质结果表明其最适pH值为6．5,最适反应温度为55℃;Na^ 、K^ 、Ca^2 、Mg^2 等离子对酶有激活作用,而Zn^2 、Ag^ 、Fe^3 和Cu^2 离子则抑制酶的活性。     

链霉菌来源D-甘露糖异构酶的性质及其在制备D-甘露糖中的应用

[背景]D-甘露糖具有多种功能活性,在食品、医药、饲料等行业应用广泛.D-甘露糖异构酶可以催化D-果糖与D-甘露糖之间的相互转化,在D-甘露糖的酶法制备中具有应用潜力.[目的]克隆一个链霉菌(Streptomyces sp.)来源的D-甘露糖异构酶基因(sssMIaseA)并在大肠杆菌中表达,研究其酶学性质,并用于制备...     

Preparation and some properties of cholesterol oxidase from Rhodococcus sp. R14-2

Rhodococcus sp. R_14-2, isolated from Chinese Jin-hua ham, produces a novel extracellular cholesterol oxidase (COX). The enzyme was extracted from fermentation broth and purified 53.1-fold based on specific activity. The purified enzyme shows a single polypeptide band on SDS-PAGE with an estimated molecular weight of about 60 kDa, and has a pI of 8.5. The first 10 amino acid residues of the NH₂-terminal sequence of the enzyme are A-P-P-V-A-S-C-R-Y-C, which differs from other known COXs. The enzyme is stable over a rather wide pH range of 4.0–10.0. The optimum pH and temperature of the COX are pH 7.0 and 50°C, respectively. The COX rapidly oxidizes 3β-hydroxysteroids such as cholesterol and phytosterols, but is inert toward 3α-hydroxysteroids. Thus, the presence of a 3β-hydroxyl group appears to be essential for substrate activity. The Michaelis constant (Km) for cholesterol is estimated at 55 μM; the COX activity was markedly inhibited by metal ions such as Hg²⁺ and Fe³⁺ and inhibitors such as p-chloromercuric benzoate, mercaptoethanol and fenpropimorph. Inhibition caused by p-chloromercuric benzoate, mercuric chloride, or silver nitrate was almost completely prevented by the addition of glutathione. These suggests that -SH groups may be involved in the catalytic activity of the present COX.     

Nucleotide sequence of the gene for cholesterol oxidase from a Streptomyces sp.

The nucleotide sequence of a 2.1-kilobase-pair fragment containing the Streptomyces choA gene, which codes a secreted cholesterol oxidase, was determined. A single open reading frame encodes a mature cholesterol oxidase of 504 amino acids, with a calculated Mr of 54,913. The leader peptides extend over 42 amino acids and have the characteristics of a signal sequence, including basic amino acids near the amino terminus and a hydrophobic core near the signal cleavage site. Analyses of the total amino acid composition and amino acid sequencing of the first 21 amino acids from the N terminus of the purified extracellular enzyme agree with the values deduced from nucleotide sequencing data.     

Effect of amino acids on production of xylanase and pectinase from Streptomyces sp. QG-11-3

Xylanase and pectinase production by Streptomyces sp. QG-11-3 was stimulated by DL-norleucine, L-leucine, DL-isoleucine, L-lysine monohydrochloride and DL--phenylalanine by up to 3.72- and 2.78-fold, respectively, whereas the combination of DL-norleucine, L-leucine and DL-isoleucine synergistically stimulated the xylanase and pectinase production by up to 6.72- and 5.62-fold, respectively. Glycine, DL-norvaline, DL-methionine, and DL-aspartic acid showed no significant stimulatory effect on enzyme production.     

Enhanced production of a thermostable xylanase from Streptomyces sp. QG-11-3 and its application in biobleaching of eucalyptus kraft pulp

A thermostable and cellulase-free xylanase has been produced from Streptomyces sp. QG-11-3 in solid substrate fermentation using wheat bran and eucalyptus kraft pulp as the prime solid substrates. The maximum xylanase yield obtained using these two substrates were 2360 U/g and 1200 U/g dry solid substrate at substrate:moisture ratios of 1:3 and 1:2.5, respectively. In immobilized cell system using polyurethane foam (PUF) and three nonwoven fabrics, namely, polyester, silk, and cotton, the xylanase yields were enhanced by 2.5-fold (203 U/ml), 1.91-fold (155 U/ml), 1.54-fold (125 U/ml), and 1.47-fold (119 U/ml), respectively, compared to the xylanase yield in liquid-batch fermentation (81 U/ml). In the biobleaching experiments, the xylanase dose of 3.5 U/g moisture free pulp exhibited the optimum bleach boosting of eucalyptus kraft pulp at pH 8.5 and 50 degrees C after 2 h of treatment. When xylanase treated pulp was subsequently treated with 4.5% chlorine, it resulted in reduction of kappa number by 25%, enhanced the brightness (%ISO) by 20% and improved the pulp properties such as tensile strength and burst factor by up to 63% and 8%, respectively.     

Expression of Streptomyces sp. cholesterol oxidase in Lactobacillus casei

Summary Three strains of Lactobacillus casei were electrotransformed with pNCO937, an 8.1-kb recombinant plasmid carrier of a Streptomyces sp. cholesterol oxidase gene. Transformation frequency was generally low and strain-dependent, ranging from 6 to 40 transformants/g DNA. L. casei transformants stably maintained pNCO937 with no indication of deletion mutational events. Transformants produced active cholesterol oxidase and sonicated cells formed 4-cholesten-3-one from both free and lipoprotein-bound cholesterol. L. casei shows promise as a host suitable for studying heterologous gene expression in lactobacilli. Correspondence to: G. A. Somkuti